Assay of neural differentiation transcripts using genomic techniques: acetylcholinesterase transcripts in aganglionosis coli in humans

Purves, Tertia Deborah

Assay of neural differentiation transcripts using genomic techniques: acetylcholinesterase transcripts in aganglionosis coli in humans

Thesis / Dissertation

1997

Publisher

University of Cape Town

Department

Department of Medicine

Faculty

Faculty of Health Sciences

Abstract

Application of mRNA analysis technologies, as a technique for protein detection, for the provision of information regarding gene expression and cellular function is an important new diagnostic tool and enhances the information gained from immuno-and enzymehistochemical detection of proteins. A unique opportunity to apply genomic technologies to an unresolved clinical problem was provided by the availability of colonic material from patients with Hirschsprung's disease (HD). HD is a relatively common condition (1:5000 live births) characterised by aganglionosis of the myenteric and submucosal plexuses of the distal colon. The pathophysiology of the disease is unknown but it is thought to arise due to a failure of neural crest cells to migrate to the distal colon during embryogenesis. It is characterised by a striking quantity of AChE enzyme activity in the distal aganglionic colon compared to much lower levels in proximal ganglionic colon as well as in normal colonic tissue. This difference in AChE enzyme activity was investigated using the mRNA analysis technologies of quantitative RT-PCR and RNA primed in situ labelling (PRINS) to detect the specific mRNA transcript, E4,6 AChE mRNA, responsible for the AChE protein. Due to the striking AChE picture there was a good chance of detecting the cells involved such as primitive or undifferentiated ganglion cells, if the corresponding mRNA was made locally. Cell lines were used as initial test vehicles to validate the use of these techniques. Quantitation of the specific transcript using a specially constructed internal standard in the RT-PCR reaction proved both reliable and reproducible and indicated a higher level of expression in K562 cells than N2a cells. The transcript was localised using the PRINS reaction in K562 cells but not in N2a cells indicating a lower limit of mRNA for the technique to be applicable. Analysis of colonic tissue from HD patients proved to be more difficult and the E4,6 AChE mRNA was quantitated in only 1 out of 4 patients who expressed higher levels of mRNA in aganglionic than ganglionic colonic tissue, although in situ localisation of this transcript in colonic tissue was unsuccessful. These techniques require much optimisation and must take into account the half-life of mRNA, mRNA stability and post-translational modification of proteins. However, this lack of detection of AChE mRNA in the colon supports the contention that the origin of the AChE protein is extrinsic to the colon, originating of necessity in the perikaryon of neurons located outside the colon. The technique of using specific mRNA detection to widen the understanding of the genetic programme of cells is feasible and should provide a valuable new tool in the diagnostic pathology laboratory once all the problems have been resolved.

Keywords

aganglionosis coli

mRNA

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